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NMRstudyofcomplexesbetweenlowmolecular mass
inhibitors andtheWestNilevirusNS2B–NS3 protease
Xun-Cheng Su
1
, Kiyoshi Ozawa
1
, Hiromasa Yagi
1
, Siew P. Lim
2
, Daying Wen
2
,
Dariusz Ekonomiuk
3
, Danzhi Huang
3
, Thomas H. Keller
2
, Sebastian Sonntag
2
,
Amedeo Caflisch
3
, Subhash G. Vasudevan
2,
* and Gottfried Otting
1
1 Research School of Chemistry, Australian National University, Canberra, Australia
2 Novartis Institute for Tropical Diseases, Singapore
3 Department of Biochemistry, University of Zu
¨
rich, Switzerland
Introduction
West Nilevirus (WNV) encephalitis is a mosquito-
borne disease that infects mainly birds, but also
animals and humans. It occurs in Africa, Europe and
Asia and, since 1999, has also been spreading in North
America, causing several thousand cases per year, with
a fatality rate of 5%, as reported by the US Depart-
ment of Health [1].
WNV is a member ofthe flavivirus genus along with
yellow fever virus, dengue virusand Japanese encepha-
litis virus, all of which cause human diseases. There is
no vaccine or specific antiviral therapy currently in
existence for WNV encephalitis in humans. During
infection, the flavivirus RNA genome is translated
into a polyprotein, which is cleaved into several
Keywords
drug development; inhibitors; NMR
spectroscopy; NS2B–NS3 protease; West
Nile virus
Correspondence
G. Otting, Research School of Chemistry,
Australian National University, Canberra,
ACT 0200, Australia
Fax: +61 2 612 50750
Tel: +61 2 612 56507
E-mail: go@rsc.anu.edu.au
*Present address
Program in Emerging Infectious Diseases,
Duke-NUS Graduate Medical School,
Singapore
Note
Xun-Cheng Su and Kiyoshi Ozawa
contributed equally to this work
(Received 28 February 2009, revised 9 April
2009, accepted 4 June 2009)
doi:10.1111/j.1742-4658.2009.07132.x
The two-component NS2B–NS3proteaseofWestNilevirus is essential for
its replication and presents an attractive target for drug development. Here,
we describe protocols for the high-yield expression of stable isotope-
labelled samples in vivo and in vitro. We also describe the use of NMR
spectroscopy to determine the binding mode of new low molecular
mass inhibitorsoftheWestNilevirusNS2B–NS3protease which were
discovered using high-throughput in vitro screening. Binding to the sub-
strate-binding sites S1 and S3 is confirmed by intermolecular NOEs and
comparison with the binding mode of a previously identified low molecular
mass inhibitor. Our results show that all these inhibitors act by occupying
the substrate-binding site oftheprotease rather than by an allosteric mech-
anism. In addition, the NS2B polypeptide chain was found to be positioned
near the substrate-binding site, as observed previously in crystal structures
of theprotease in complex with peptide inhibitors or bovine pancreatic
trypsin inhibitor. This indicates that the new lowmolecularmass com-
pounds, although inhibiting the protease, also promote the proteolytically
active conformation of NS2B, which is very different from the crystal
structure ofthe protein without inhibitor.
Abbreviations
BPTI, bovine pancreatic trypsin inhibitor; Bz-nKRR-H, benzoyl-norleucine-lysine-arginine-arginine-aldehyde; HTS, high-throughput screen;
WNV, WestNile virus.
4244 FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS
components. Nonstructural protein 3 (NS3) is responsi-
ble for proteolysis ofthe polyprotein through its serine
protease N-terminal domain (NS3pro), in conjunction
with a segment of 40 residues from the NS2B protein
acting as a co-factor. NS3 is essential for viral replica-
tion and therefore presents an attractive drug target.
The C-terminal two-thirds of NS3, which contain a
nucleotide triphosphatase, an RNA triphosphatase and
a helicase, have been shown to have little influence on
protease activity [2], although the 3D structure of
the full-length dengue virus DENV-4 NS3 protease–
helicase suggests that theprotease domain assists the
binding of nucleotides to the helicase and may also
participate in RNA unwinding [3].
Crystal structures of WNV NS2B–NS3pro have
been reported in the absence of inhibitor [4] and in the
presence of peptide inhibitors [5,6] or bovine pancre-
atic trypsin inhibitor (BPTI) [4]. In the absence of
inhibitor, the structure shows the b-hairpin of NS2B
positioned far (almost 40 A
˚
) from the active site.
Because the C-terminal residues of NS2B are not only
essential for full catalytic activity of WNV NS2B–
NS3pro [7,8], but are also found near the active site
in the structures with peptide inhibitorsand BPTI,
the proteolytically most active conformations are
thought to be represented by the structures observed
with inhibitors rather than the one without inhibitor.
The function oftheprotease is preserved in a 28 kDa
construct in which NS2B and NS3pro are fused via a
Gly
4
–Ser–Gly
4
linker (Fig. 2) [2,9].
A number oflowmolecularmass nonpeptidic inhibi-
tors have been generated in hit-to-lead activities fol-
lowing a high-throughput screen (HTS) directed
against dengue virusNS2B–NS3protease (C. Bodenre-
ider et al., manuscript in preparation). Because of the
high sequence homology between dengue virus and
WNV, many ofthe compounds found to inhibit the
dengue virusprotease also inhibited WNV protease,
albeit with different affinities (C. Bodenreider et al.,
manuscript in preparation). Figure 1 shows three of
the inhibitors found. Compounds 1 and 2 originated
from the HTS, whereas compound 3 was discovered
using the crystal structure of WNV NS2B–NS3pro
with bound tetrapeptide [5] in an in silico screening
approach [10]. Compounds 1 and 2 showed inhibition
constants in thelow micromolar range, but no related
compounds could be found with inhibition constants
below 1 lm (C. Bodenreider et al., manuscript in prep-
aration).
The results of two other published HTS efforts
confirmed that discovery of high-affinity inhibitors for
WNV NS2B–NS3pro is nontrivial. In one study,
competitive inhibitors with an inhibition constant of
3 lm were found and their binding to WNV NS2B–
NS3pro modelled [11]. In another, noncompetitive
inhibitors with IC
50
values of 0.1 lm were found, but
these were prone to hydrolysis with deactivation half-
lives of 1–2 h. The latter are thought to bind to
NS3pro, displacing the C-terminal b-hairpin of NS2B
from NS3pro [12]. HTS campaigns against the WNV
replicon, in which the target protein is unknown, also
failed to discover nonpeptidic inhibitors with inhibi-
tory activities much below 1 lm [13,14], with an EC
50
value of 0.85 lm being reported for the most active
compound [15].
In order to improve our understanding ofthe action
of compounds 1–3 against WNV NS2B–NS3pro, struc-
tural information about their binding modes must be
obtained. Despite many efforts, however, no crystal
structure oftheprotease could be determined in com-
plex with compounds 1–3 or any other low molecular
mass inhibitor. In view ofthe ability of NS2B to
undergo a large structural change between proteolyti-
cally deactivated and fully active states, as observed in
crystal structures [4,5], competitive inhibition may con-
ceivably be achieved by binding to an allosteric site
rather than to the active site. We therefore turned to
solution NMR spectroscopy to identify the binding
sites of 1–3 to WNV NS2B–NS3pro.
We have previously described a model of 3 bound
to WNV NS2B–NS3pro, obtained by automatic
computational docking, which is in agreement with the
Fig. 1. Synthetic inhibitors 1–3 of WNV NS2B–NS3pro studied.
Individual atoms are numbered as reference for NMR resonance
assignments.
X C. Su et al. NMR analysis oftheWestNilevirus protease
FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS 4245
intermolecular NOEs reported here [10]. Ekonomiuk
et al. [10] also presented the dissociation constant of 3
measured by NMR and, as additional proof for bind-
ing of 3 to the substrate-binding site, demonstrated
changes in cross-peak positions for residues lining the
substrate-binding site, without discussing the complete
resonance assignment.
In the following, we report protocols for the expres-
sion of isotope-labelled WNV NS2B–NS3pro in high
yields in Escherichia coli in vivo and by cell-free syn-
thesis, the first virtually complete assignments of the
15
N-HSQC spectrum, structure analysis of WNV
NS2B–NS3pro with bound inhibitor, and identification
of intermolecular NOEs betweentheinhibitorsand the
protease.
Results
Sample preparation
The original construct of NS2B–NS3pro (construct 1,
Fig. 2) was toxic to E. coli, leading to cell lysis on
plates prepared with rich media as well as in large-
scale preparations. Improved protein yields were
obtained by a modified protocol, where E. coli colonies
grown on M9 media plates were selected prior to
large-scale expression. In this way, 9.3 mg of purified
uniformly
15
N ⁄
13
C-labelled protein were obtained per
litre of a
15
N ⁄
13
C-labelled rich medium (induction by
isopropyl b-d-thiogalactoside), whereas an autoinduc-
tion protocol [16] yielded as much as 59 mg of purified
15
N-labelled protein per litre of cell culture (Materials
and methods).
Construct 1 equally produced hardly any protein in
our cell-free protein synthesis system [17,18]. This
problem was overcome by construct 2 which starts
with the first six codons from T7 gene 10 and which
expresses well in cell-free systems. A clone in a high-
copy number T7 plasmid [19] facilitated the prepara-
tion of large quantities of DNA required for the
cell-free synthesis. Typical yields were close to 1 mg of
purified protein per mL of cell-free reaction mixture.
Although acceptable
15
N-HSQC spectra could be
recorded without purification ofthe protein [20,21],
complex formation with theinhibitors required puri-
fied protein because compounds 1 and 2 also bound to
components ofthe cell-free mixture.
The NS2B–NS3pro construct 1 in Fig. 2 was suscepti-
ble to gradual self-cleavage by theprotease at two sites,
following the first glycine in the linker after Lys96
NS2B
and Lys15
NS3
(Fig. 2) [5,22], resulting in release of the
intermittent peptide from the protein. Because variable
extents of cleavage led to sample heterogeneity, later
work employed the mutant Lys96
NS2B
fi Ala (con-
struct 3) which prevented cleavage at either site [23]. The
K96A mutant turned out to be much less toxic to
E. coli, producing high yields even when overexpression
was induced by isopropyl b-d-thiogalactoside. The
K96A mutant retained full proteolytic activity in the
assay used (C. Bodenreider et al., manuscript in prepa-
ration) to measure the inhibition constant of different
ligands (data not shown).
Inhibitor binding monitored by NMR
spectroscopy
In the absence of inhibitors, assignment ofthe NMR
resonances for WNV NS2B–NS3pro was difficult
because many signals were broadened beyond detec-
tion andthe spectral resolution was poor (Fig. 3A).
Over 100 different compounds that had been suggested
by high-throughput docking calculations with a large
library of molecules [10] or had appeared as hits in the
in vitro high-throughput screens were tested for bind-
ing to WNV NS2B–NS3pro by NMR spectroscopy
using
15
N-labelled protein. 1D
1
N NMR spectra were
used to assess any line broadening experienced by the
low molecularmass compounds and
15
N-HSQC spec-
tra were recorded to detect responses in the protein.
Most ofthe compounds showed broad lines in the
presence of protein without noticeably changing the
15
N-HSQC spectrum. This situation was interpreted as
nonspecific binding. Other compounds were barely sol-
uble in water. Compounds 1 and 2, however, improved
the
15
N-HSQC spectra ofthe protein dramatically
in a manner similar to compound 3. In addition to
Fig. 2. Amino acid sequence ofthe WNV NS2B–NS3pro constructs used. In addition to the sequence shown, constructs contained the
N-terminal sequences MGSSHHHHHHSSGLVPRGSHM (construct 1) or MASMTGHHHHHH (construct 2; Materials and methods). A third
construct (construct 3) contained the mutation Lys96
NS2B
fi Ala with N-terminal MASMTGHHHHHH peptide [WNV NS2B–NS3pro(K96A)].
All constructs ended at residue 187 of NS3. Vertical lines identify two autocatalyic cleavage sites [23]. The K96A mutation prevents
self-cleavage at either site. Residues without backbone resonance assignments (disregarding proline) are highlighted in orange.
NMR analysis oftheWestNilevirusprotease X C. Su et al.
4246 FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS
improved spectral dispersion, the
15
N-HSQC spectra
of thecomplexes with 2 and 3 (Fig. S1) showed
marked similarities, indicating that both compounds
stabilize the same structure ofthe enzyme.
Compound 1 originated from the in vitro screen (C.
Bodenreider et al., manuscript in preparation). It was
the first found to improve theNMR spectrum of
WNV NS2B–NS3pro in a manner very similar to the
inhibitor benzoyl-norleucine-lysine-arginine-arginine-
aldehyde (Bz-nKRR-H) [24], which has been used for
crystallization [5]. Hence, the first resonance assign-
ments oftheprotease by 3D NMR spectroscopy were
performed using the complex with 1. Compound 2 was
designed to improve the solubility of 1 and lift its two-
fold symmetry in order to facilitate the assignment
of intermolecular NOEs. 2 bound to WNV NS2B–
NS3pro with similar affinity to 1 (IC
50
of 11 versus
25 lm) (C. Bodenreider et al., manuscript in prepara-
tion). Compound 3 inhibited WNV NS2B–NS3 by
35% when tested at 25 lm and had a K
d
value of
40 lm as measured by NMR [10].
Similar to 3 [10], as 1 or 2 were added to the enzyme
some of the
15
N-HSQC peaks shifted, indicative of
chemical shift averaging by chemical exchange on a
time scale of tens of milliseconds, whereas others
appeared at new positions, as expected for slow
exchange in the limit of large chemical shift differences
between the free and complexed protein (Fig. S2). The
15
N-HSQC spectra did not change significantly when
the inhibitors were used in excess.
Resonance assignments
The quality of the
15
N-HSQC spectra obtained in the
presence of 1, 2 or 3 was sufficient for sequential reso-
nance assignments using conventional triple-resonance
3D NMR experiments. NMR spectra of NS2B–
NS3pro and NS2B–NS3pro(K96A) were closely
similar, as expected for a point mutation in a mobile
segment ofthe polypeptide chain. Increased mobility
of the segment surrounding residue 96 in NS2B had
been suggested by the absence of electron density for
the linker peptide between NS2B and NS3 following
Asp90 in the crystal structure with BPTI [4] and was
confirmed by narrow NMR line shapes.
The resonances ofthe complex with 1 were assigned
using NS2B–NS3pro, whereas the 3D NMR experi-
ments ofthecomplexes with 2 and 3 employed the
WNV NS2B–NS3pro(K96A) mutant. The resonance
assignments ofthecomplexes with 1 and 3 were sup-
ported by combinatorial
15
N-labelling (Fig. S3). The
assignments ofthe backbone amide cross-peaks are
shown in Fig. S1. Resonance assignments were
obtained for the backbone amides ofthe segments
comprising residues 50–96 of NS2B and 17–187 of
NS3pro, with the exception of prolines and a few resi-
dues with very broad amide peaks. The resonances of
the peptide connecting NS2B and NS3pro appeared at
chemical shifts characteristic of random coil confor-
mation and were not assigned.
Conformation of WNV NS2B–NS3pro induced by
inhibitors
NOEs between NS2B and NS3pro observed for the
complex with 2 showed that NS2B docks to NS3pro
as in the crystal structures with peptidic inhibitors
(Table 1) [4–6]. Furthermore, the similarity of the
backbone amide chemical shifts seen in complexes with
1, 2 and 3 (Fig. S1) indicated that NS2B assumes the
same conformation in the presence of any ofthe three
compounds. The crystal structures of NS2B–NS3pro
A
B
Fig. 3.
15
N-HSQC spectra of WNV NS2B–NS3pro(K96A) in the
absence and presence of inhibitor 2 at 25 °C. The samples con-
tained 0.9 m
M protein in 90% H
2
O ⁄ 10% D
2
O containing 20 mM
Hepes buffer (pH 7.0) and 2 mM dithiothreitol. The complex with 2
was prepared by adding 15 lL of 100 m
M solutions of inhibitor in
d
6
-dimethylsulfoxide to the protein solution. The spectra were
recorded at a
1
N NMR frequency of 800 MHz. (A)
15
N-HSQC spec-
trum in the absence of inhibitor. (B)
15
N-HSQC spectrum in the
presence of compound 2 (3 m
M).
X C. Su et al. NMR analysis oftheWestNilevirus protease
FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS 4247
in complex with peptide inhibitors or BPTI [4–6]
are thus suitable starting points for modelling the
complexes with thelowmolecularmassinhibitors of
this study.
Inhibitor binding sites
Because theNMR spectra oftheprotease complexes
with 1 and 2 were very similar, both compounds must
bind in the same way. Therefore, we only studied the
binding ofthe nonsymmetric and more soluble com-
pound 2 using intermolecular NOEs. In the 1 : 1 com-
plex with the protease, the proton resonances of the
phthalazine ring of 2 were too broad to be observable.
(1 behaved in the same way.) Therefore, we used 2 in
an approximately three-fold excess over theprotease in
order to measure intermolecular NOEs. The maximal
solubility of 2 in water was 3mm, but aggregation
occurred at much lower concentrations. Thus, even at
0.3 mm, theNMR line widths of 2 were broader than
expected for a monomeric compound (Fig. S4).
Furthermore, negative intramolecular NOEs were
observed for a sample at 0.7 mm, indicating an effec-
tive molecularmassof > 500 Da. The possibility of
self-association made it harder to interpret the inter-
molecular NOEs observed betweentheproteaseand 2.
Consequently, we used the NOE data with 3 to sup-
port the assignment of intermolecular NOEs with 2.
Figure 4 shows intermolecular NOEs observed
between WNV NS2B–NS3pro(K96A) and 3. Although
most NOEs could readily be assigned, the difficulty of
obtaining complete side-chain resonance assignments
for the protein prompted us to seek additional verifica-
tion that 3 binds to the substrate-binding site of the
protease.
In the first experiment, we compared the
15
N-HSQC
spectra of WNV NS2B–NS3pro(K96A) in the presence
of 3 and in the presence ofthe Bz-nKRR-H inhibitor
used in one ofthe crystal structure determinations [5].
As expected for closely related binding sites, the spec-
Table 1. NOEs observed between NS2B and NS3pro in the pres-
ence of 2 or 3.
NS2B NS3 Distance ⁄ A
˚
a
Trp53 H
N
Thr27 H
a
3.7
Ala58 H
N
Val22 H
N
3.1
Asp59 H
a
Val22 H
N
3.6
Ser72 H
a
Gly114 H
N
2.8
Arg74 H
a
Val115 H
N
2.6
Val77 H
N
Lys117 H
N
3.3
Gly83 H
N
Lys73 H
a
2.8
a
Distance in the crystal structure with tetrapeptide inhibitor
(2FP7) [5].
Fig. 4. 2D NOESY spectrum with
13
C(x
2
) ⁄
15
N(x
2
) half-filter of WNV NS2B–
NS3pro(K96A) in complex with 3. Parame-
ters: 0.9 m
M protein and 2 mM 3 in 90%
H
2
O ⁄ 10% D
2
O containing 20 mM Tris ⁄ HCl
buffer (pH 7.2) and 2 m
M dithiothreitol,
25 °C, mixing time 120 ms, t
1max
= 34 ms,
t
2max
= 86 ms, 800 MHz
1
N NMR
frequency. Intermolecular NOEs with the
aromatic ring protons of 3 are marked with
their assignments. Several ofthe NOEs are
also observed with the methyl groups of 3
at 2.3 p.p.m.
NMR analysis oftheWestNilevirusprotease X C. Su et al.
4248 FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS
tra were very similar except for chemical shift changes
for some ofthe residues lining the substrate-binding
site (Fig. S5).
In another experiment, selectively
15
N-Gly-labelled
samples of WNV NS2B–NS3pro were prepared of the
wild-type protein andthe Gly151Ala mutant. Gly151
is located in close proximity to the active-site histidine
residue and mutation to alanine should interfere with
both enzyme activity and with inhibitors that target
the substrate-binding site. Indeed, the G151A mutant
was inactive in the enzymatic assay [25] and unable to
bind 3 (Fig. S6).
Having established that compound 3 occupies the
substrate-binding site, we used the INPHARMA strat-
egy [26] to verify that compound 2 is also residing in
the substrate-binding site. A NOESY spectrum of 2
and 3 in the presence of a small quantity of protease
revealed an intermolecular cross-peak between the
methyl group of 3 andthe phthalazine ring of 2,as
expected for an overlapping binding site (Fig. 5).
Table 2 compiles the intermolecular NOEs observed
with 2 and 3. The NOEs with Ile155 were most readily
assigned because of their characteristic chemical shifts,
whereas other NOEs were assigned using the assump-
tion that theprotease fold was that observed in the
crystal structures with peptide inhibitors. The fact that
all intermolecular NOEs observed with the aromatic
ring proton of 3 were also observed with the methyl
group was, in most cases, probably a consequence of
spin-diffusion. Relaxation during the half-filter delays
and the twofold symmetry of 3 further impeded accu-
rate distance measurements.
The data show that both inhibitors are in proximity
of Thr132 and Ile155. There are, however, also signifi-
cant differences betweenthe binding modes ofthe two
compounds. For example, 3 contacts the side chain of
His51 in the active site, whereas no equivalent interac-
tion could be found for 2. No intermolecular NOE
with NS2B could be observed because ofthe difficulty
of observing proton resonances of amino and guanidi-
nium groups.
Model building
Docking of compound 2 was performed automatically
by daim ⁄ seed ⁄ ffld [27–31] using the PDB coordinate
set 2FP7 [5], as described previously for 3 [10]. For
each compound, a total of 50 poses was kept upon
clustering. The pose which best satisfied the inter-
molecular NOEs (Table 2) was selected as the final
model. Not all cross-peaks observed for 2 (Table 2)
could be explained as direct NOEs with the protease.
This may be because of spin-diffusion during the mix-
ing time ofthe NOESY experiment, movements of the
ligand in the binding pocket or differences in side-
chain orientations betweenthe crystal and solution
Fig. 5. 2D NOESY spectrum of 0.6 mM 2 and 0.5 mM 3 in the
presence of 0.03 m
M WNV NS2B–NS3pro(K96A) in D
2
Oat25°C.
Under these conditions, the signals of 2 were sufficiently narrow to
be observable (Fig. S4C). Other parameters: mixing time 150 ms,
t
1max
= 35 ms, t
2max
= 71 ms. The cross-peak between 3 H3 and 2
H6 or H6¢ is assigned as well as the intramolecular NOE between
3 H3 and H1.
Table 2. Intermolecular NOEs betweenWestNilevirus (WNV)
NS2B–NS3pro(K96A) andinhibitors 2 and 3.
Protons of WNV NS3pro Compound 2
a
Compound 3
His51 H
d2
H1 and CH
3
Tyr130 H
d
H6 ⁄ H6¢
Thr132 C
!2
H
3
H6 ⁄ H6¢ H1 and CH
3
Thr132 H
a
H1 and CH
3
Thr134 C
!2
H
3
H6 ⁄ H6¢
Tyr150 H
d
H6 ⁄ H6¢
Asn152 C
b
H
2
H6 ⁄ H6¢
Gly153 H
N
H1
Val154 C
!
H
3
H1, H2, H5 ⁄ H5¢ H1 and CH
3
Ile155 C
d1
H
3
H1, H2, H3, H4 H1 and CH
3
Tyr161 H
d
H1 and CH
3
a
NOEs identified in Fig. 6 are underlined.
X C. Su et al. NMR analysis oftheWestNilevirus protease
FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS 4249
structure. [For example, the side chain of Ile155
is differently oriented in the structure with BPTI
(v
1
= )66°) [4] than in the structure used for Fig. 6
(v
1
= )180°) [5], andthe intermolecular NOEs
observed with Ile155 are in much better agreement
with v
1
= )180° than v
1
= )66°.] In the case of
aggregation-prone compound 2, binding of more than
a single molecule may have confounded the interpreta-
tion of intermolecular NOEs. Nonetheless, the model
in Fig. 6A satisfies most NOEs. It places the positively
charged cyclic amidine group near the negatively
charged side chain of Asp129 which interacts with the
positively charged side chain ofthe P1 residues of
Bz-nKRR-H [5] and BPTI [4]. The primary amino
group of 2 points towards the C-terminal b-hairpin of
NS2B which carries three aspartate residues in a row
in positions 80–82. Although 2 belongs to a different
class of compounds than 3, the binding modes of both
compounds are not dissimilar (Fig. 6).
Discussion
Competitive inhibition is usually accepted as strong
indication that the binding sites of two inhibitors are
at least partially overlapping. In the case ofthe WNV
NS2B–NS3 protease, the C-terminal b-hairpin of
NS2B is essential for catalytic activity, but has been
found far away from the substrate-binding site in the
absence of inhibitor [4]. In addition, the substrate-
binding site changes significantly between the
A
B
Fig. 6. Stereoviews of models of 2 and 3
bound to WNV NS2B–NS3pro. The protein
structure is that by Erbel et al. [5], with
NS2B drawn as a grey ribbon. Heavy atom
representations of 2 and 3 are drawn in
black. The side chains of residues for
which intermolecular NOEs are reported in
Table 2 are shown in a stick representation.
(A) Complex with 2. Selected intermolecular
NOEs (Table 2) are highlighted with
magenta lines. (B) Complex with 3 reported
in Ekonomiuk et al. [10].
NMR analysis oftheWestNilevirusprotease X C. Su et al.
4250 FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS
structures with and without inhibitor, so that competi-
tive inhibition may conceivably be achieved by binding
to a site that prevents NS2B from correct association
with the substrate-binding site. In this situation, NMR
spectroscopy provides an important tool for the identi-
fication ofthe inhibitor binding site.
No sequence-specific NMR resonance assignments
have been reported for the WNV NS2B–NS3 protease.
The poor quality oftheNMR spectrum of WNV
NS2B–NS3pro in the absence ofinhibitors is reminis-
cent ofthe situation in the homologous NS2B–NS3pro
construct from dengue virus type 2, in which selec-
tively
15
N ⁄
13
C-labelled samples show a great variation
in NMR line-width, prohibiting conventional assign-
ment strategies by multidimensional NMR spectro-
scopy [32]. The dramatic improvement in spectral
quality observed upon formation ofcomplexes with
our inhibitors is readily explained by a shift in confor-
mational exchange equilibria towards a single con-
former. NOEs between NS2B and NS3 indicate that
this conformer is related to the conformation observed
in the crystal structures ofthe complex with peptidic
inhibitors [4–6], in which the C-terminal b-hairpin of
NS2B is positioned near the substrate-binding site
rather than far away as in the crystal structure in the
absence of inhibitor [4]. We were able to obtain this
result without optimized engineering ofthe NS2B part
that had been required to obtain an acceptable NMR
spectrum ofthe closely related dengue virus NS2B–
NS3 protease [33].
The NMR data clearly show that the small synthetic
inhibitors 1–3 bind to the substrate-binding site of
WNV NS2B–NS3pro. Competitive inhibition with
established peptide inhibitors is thus effected by direct
competition rather than by indirect competition via an
allosteric inactivation mechanism. Considering the
apparent ease with which the C-terminal b-hairpin of
NS2B is brought into the vicinity ofthe active site,
our results indicate that the crystal structures of the
protease–peptide complexes are valid starting points
for the search for lowmolecularmass inhibitors.
Indeed, compound 3 is the first inhibitor of WNV
NS2B–NS3pro that has been discovered by a computer
search using the crystal structure with a tetrapeptide
inhibitor as a template [5,10]. An important implica-
tion is that the only available crystal structure of the
corresponding dengue virusprotease [5] is not a suit-
able starting point, because it positions the C-terminal
b-hairpin of NS2B far from the substrate-binding site.
Although compounds 1–3 induce a more uniform
structure of WNV NS2B–NS3pro, they are not able to
suppress all conformational exchange. For example,
we could not assign the backbone amides of Thr132,
Gly133 and Gly151 even in the presence of 1, 2 or 3,
and the backbone resonances of neighbouring residues
were broad. All three residues line the substrate-bind-
ing pocket. In order to find improved inhibitors, it is
thus relevant to explore the conformational space of
the protease in a molecular dynamics simulation rather
than relying exclusively on the structures observed
in the solid state. Intriguingly, the Thr132–Gly133
peptide bond was found to flip spontaneously in the
course of two 80-ns and one 40-ns molecular dynamics
simulations performed recently [34]. A flip of this
peptide bond also presents the main difference in
backbone conformation ofthe substrate-binding site
between the crystal structures 2IJO and 2FP7 [4].
The Gly
4
–Ser–Gly
4
linker connecting NS2B and
NS3pro is highly flexible in solution because the corre-
sponding signals appeared in an intense cluster of peaks
at a chemical shift characteristic of a random coil pep-
tide chain. Structural variability of these residues has
initially been suggested by the absence of electron den-
sity for the linker residues andthe C-terminal residues
of NS2B following Asn89 in the WNV NS2B–
NS3pro(K96A) mutant in complex with BPTI [4]. Also,
the recent structure oftheprotease in complex with a
tripeptide inhibitor misses electron density for, respec-
tively, three or all ofthe residues ofthe Gly
4
–Ser–Gly
4
linker in the two conformers reported [6]. The high
mobility observed by NMR for the peptide linker in
solution provides a firm explanation for the finding that
the covalent linkage between NS2B and NS3 does not
restrain the function oftheprotease [2,9].
In conclusion, compounds 1 and 2 target the sub-
strate-binding site ofthe WNV NS2B–NS3 protease.
Their binding site overlaps with that of compound 3
(Fig. 6). Remarkably, even these small, nonpeptide
inhibitors can stabilize the conformation of NS2B
observed in crystal structures with peptides. This result
provides crucial validation for the use of computa-
tional approaches that start from the crystal structures
obtained with peptide inhibitors [10]. It also underpins
the success of further computations that, by taking
into account the conformations sampled by molecu-
lar dynamics simulations, led to nonpeptidic lead
compounds with low-micromolar affinity [35].
Materials and methods
Materials
Compounds 1 and 2 were synthesized in-house. Compound
3 was obtained from Maybridge (Tintagel, UK) (Cat#
S01870SC). Spectra 9 (
13
C,
15
N) media was obtained from
Spectra Stable Isotopes (Columbia, MD, USA).
15
NH
4
Cl,
X C. Su et al. NMR analysis oftheWestNilevirus protease
FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS 4251
13
C ⁄
15
N-Silantes (OD2) media,
15
N-glycine,
13
C ⁄
15
N-tyro-
sine and
13
C ⁄
15
N-phenylalanine were purchased from Cam-
bridge Isotope Laboratories (Andover, MA, USA). E. coli
strains Rosetta::kDE3 ⁄ pRARE and BL21 Star::kDE3
were obtained from Novagen (Gibbstown, NJ, USA) and
Invitrogen (Carlsbad, CA, USA), respectively. Synthetic
oligonucleotides were purchased from GeneWorks (Hind-
marsh, Australia). Sequences of oligonucleotides used are
listed in the Supporting Information. Vent DNA polymer-
ase and Phusion DNA polymerase were obtained from
New England BioLabs (Ipswich, MA, USA). Qiaquick
PCR purification and Qiaquick gel extraction kits were
purchased from Qiagen (Hilden, Germany).
Preparation of uniformly
15
N-labelled WNV
NS2B–NS3pro
The E. coli strain Rosetta::k DE3 ⁄ pRARE was transformed
with the plasmid pET15b–WNV CF40GlyNS3pro187 (con-
struct 1 of Fig. 2) [5] on Luria–Bertani plates containing
100 lgÆmL
–1
ampicillin and 50 lgÆmL
–1
chloramphenicol. A
single transformant colony (10
8
cells) was diluted with
Luria–Bertani media to 10
7
cells in 1 mL of Luria–
Bertani and 100 lL batches ofthe diluted cells were plated
on 15 M9 minimal media plates, containing 5 mm glucose,
0.2% (w ⁄ v) glycerol, 100 lgÆmL
–1
ampicillin and
50 lgÆmL
–1
chloramphenicol. Following growth for 2 days
at 37 °C, the colonies were collected and resuspended in
small volumes of M9 media. Approximately 100 D
595
units
of cells were used to inoculate 500 mL of
15
N-autoinduc-
tion media containing 0.5 gÆL
–1 15
NH
4
Cl, 100 lgÆmL
)1
ampicillin and 50 lgÆmL
)1
chloramphenicol [16]. Four con-
ical 2-L flasks, each containing 500 mL of
15
N-autoinduc-
tion cultures, were shaken at room temperature at 200 rpm
for 2 days up to an D
595
value of 5, yielding 16.6 g of
cells. The cells were suspended in 80 mL of buffer A
(50 mm Hepes, pH 7.5, 300 mm NaCl, 5% glycerol, 20 mm
imidazole) and lysed by a French press (12 000 psi, two
passes). After centrifuging the lysate at 15 000 g for 1 h,
the supernatant was filtered through a 0.45 lm Millipore
filter. The filtrate was directly loaded on a 5 mL Ni-NTA
column (Amersham Biosciences, Uppsala, Sweden). The
bound
15
N-WNV NS2B–NS3pro was eluted with an imid-
azole gradient of 20–500 mm in buffer A. The overall yield
of purified protein was 118 mg per 2 L of culture. The pro-
tein concentration was determined spectrophotometrically
at 280 nm, using a calculated e
280
value of 55 760 [36] and
the purity checked by SDS ⁄ PAGE.
For subsequent testing of different compounds by
15
N-HSQC spectra in 3 mm NMR tubes, the protein was
subdivided into over 100 batches of 200 lL each, contain-
ing 7 mgÆmL
)1
protein in NMR buffer [20 mm Hepes ⁄
KOH, pH 6.98, 90% H
2
O ⁄ 10% D
2
O, 1 mm tris(2-carboxy-
ethyl)phosphine or 2 mm dithiothreitol]. A sample was pre-
pared for each individual compound by injecting 3 lLof
100 mm solutions of compound in d
6
-dimethylsulfoxide into
200 lL of aqueous protein solution in a 3 mm NMR tube.
Preparation of uniformly
13
C/
15
N-labelled WNV
NS2B–NS3pro
13
C ⁄
15
N-labelled WNV NS2B–NS3pro was prepared using
the same protocol as for
15
N-labelled WNV NS2B–NS3pro,
except that 2 · 500 mL of
13
C ⁄
15
N-Silantes media (OD2)
were used which were supplemented with 100 lgÆmL
–1
ampicillin and 33 lgÆmL
)1
chloramphenicol. The cells were
grown at 37 °C and 200 r.p.m. for 6 h before induction
with 0.6 mm isopropyl b-d-thiogalactoside at D
595
= 0.95.
The induced cells were grown at room temperature over-
night to D
595
= 1.1, yielding 1.8 g of cells which were
suspended in 20 mL buffer A for purification as described
above. The final yield of
13
C ⁄
15
N-labelled protease was
9.3 mg in NMR buffer. The sample used for 3D NMR
experiments was 0.4 mm in protein in a 5 mm NMR tube.
Preparation of uniformly
13
C/
15
N-labelled WNV
NS2B–NS3pro(K96A)
A
13
C ⁄
15
N-labelled sample ofthe K96A mutant of WNV
NS2B–NS3pro (construct 3, Fig. 2) was prepared using the
same protocol as for
13
C ⁄
15
N-labelled WNV NS2B–NS3pro,
except that 2 · 500 mL of
13
C ⁄
15
N-Spectra 9 media was
used, which was supplemented with 100 lgÆmL
)1
ampicillin
and 50 lgÆmL
)1
chloramphenicol. Cells were grown at 37 °C
and 200 rpm for 3 h before induction with 0.6 mm isopropyl
b-d-thiogalactoside at D
595
= 1. The induced cells were
grown at room temperature overnight to D
595
= 1.9, yield-
ing 4.4 g of cells which were suspended in 50 mL buffer A
for purification on a 5 mL Ni-NTA column as described
above. Following elution from the column, the protein was
dialysed against 1 L of 50 mm Tris ⁄ HCl (pH 7.6). The
dialysate was loaded on a 7.4 mL DEAE-Toyopearl 650M
column (2.5 · 1.5 cm; Tosoh Bioscience, Montgomeryville,
PA, USA) andthe bound protease eluted by a NaCl gradient
of 0 mm to 1 m in a buffer of 50 mm Tris ⁄ HCl (pH 7.6) and
1mm dithiothreitol. The final yield of
13
C ⁄
15
N-labelled
protease was 48.4 mg in NMR buffer. NMR samples were
0.9 mm in protein.
Cell-free synthesis of WNV NS2B–NS3pro
Construct 2 (Fig. 2) was designed for optimum expression
yields in a cell-free system. Primers 1307 and 1308
(Table S1) were used to amplify theprotease gene by PCR
from the template plasmid pET15b-WNV CF40glyN-
S3pro187 using Phusion DNA polymerase. Following
digestion by NdeI and EcoRI, the PCR fragment was trans-
ferred into the corresponding site ofthe pRSET-5b vector
[19]. The resulting vector (pRSET-WNV MASMTGH
6
-
NMR analysis oftheWestNilevirusprotease X C. Su et al.
4252 FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS
CF40glyNS3pro187) was used for cell-free protein synthesis
using a cell extract from E. coli.
S30 cell extracts were prepared from the E. coli strains
Rosetta::kDE3 ⁄ pRARE and BL21 Star::kDE3 as described
previously [17,18,37], including concentration with poly-
ethylene glycol 8000 [38] and heat treatment ofthe concen-
trated extracts at 42 °C [39].
Cell-free protein synthesis was performed for 6–7 h either
using an autoinduction system with plasmid pKO1166 for
in situ production of T7 RNA polymerase [40] or using
a standard protocol with purified T7 RNA polymerase
at 37 or 30 °C [18,21]. The reactions were performed
with lgÆmL
)1
target plasmid. Site-directed mutants were
produced from 5 to 10 lgÆmL
)1
PCR-amplified DNA tem-
plates. Following cell-free synthesis, the reaction mixtures
were clarified by centrifugation (30 000 g, 1 h) at 4 °C.
Cell-free synthesis of combinatorially
15
N-labelled
WNV NS2B–NS3pro
Five sets of
15
N-combinatorially labelled samples [41,42]
of construct 2 (Fig. 2) were produced by cell-free protein
synthesis. Synthesis was performed using 1 mL reaction
mixtures for sets 1–4 and 2 mL for set 5. Set 5 was the only
reaction containing
15
N-glutamate. This set was prepared
using 100 mm potassium succinate in the reaction mixture
instead ofthe usual 208 mm potassium glutamate buffer.
Cell-free protein synthesis was performed at 37 °C for 6 h.
Following centrifugation, the supernatants were diluted with
5–10 mL of buffer A andthe proteins purified by a 1 mL
Ni-NTA column (Pharmacia) using a 20–500 mm imidazole
gradient in buffer A. The buffer ofthe samples was
exchanged to 20 mm Hepes ⁄ KOH (pH 7.0) and 1 mm tris(2-
carboxyethyl)phosphine using Millipore Ultra-4 centrifugal
filters (molecular mass cutoff 10 000), followed by concen-
tration to a final volume of 0.2 mL. D
2
O was added to a
final concentration of 10% (v ⁄ v) prior to NMR measure-
ments, resulting in a protein concentration of 50 lm.
Cell-free synthesis of
15
N-Gly labelled wild-type
and mutant WNV NS2B–NS3pro
Wild-type and mutant (Gly151Ala) samples of selectively
15
N-Gly labelled WNV NS2B–NS3pro (construct 2) were
produced by cell-free synthesis from cyclized PCR tem-
plates [32] using primers 1314, 1315 and 1131–1134
(Table S1). The synthesis was performed in 1 mL reaction
mixtures, using the same conditions and purification proto-
col as for the combinatorially labelled samples.
NMR measurements
All NMR spectra were recorded at 25 °C using Bruker 800
and 600 MHz Avance NMR spectrometers equipped
with TCI cryoprobes. Samples ofcomplexes contained
an approximately three-fold excess of inhibitor in order to
facilitate the observation of intermolecular NOEs. 3D spec-
tra recorded included HNCA, HN(CO)CA, CC(CO)NH,
(H)CCH-TOCSY and NOESY-
15
N-HSQC (mixing time
60 ms). NOESY spectra with
13
C(x
2
) ⁄
15
N(x
2
) half-filters
(mixing time 120 ms) were used to suppress intramolecular
NOEs oftheproteaseand observe intermolecular NOEs.
For unambiguous identification of intraligand NOEs, the
experiment was also recorded with a
13
C-BIRD sequence in
the middle ofthe mixing time which suppressed any NOE
from
13
C-bound protons ofthe protein. A 3D
13
C-HMQC-
NOESY spectrum with
13
C ⁄
15
N(x
2
) half-filter (mixing time
150 ms) facilitated the assignment ofthe intermolecular
NOEs by comparison with the (H)CCH-TOCSY spectrum.
The chemical shifts have been deposited in the BioMagRes-
Bank (accession number 11053).
Acknowledgements
This work was supported by the Australian Research
Council. Docking calculations were performed on the
Matterhorn computer cluster at the University of
Zu
¨
rich.
References
1 Hayes EB & Gubler DJ (2006) WestNile virus: epide-
miology and clinical features of an emerging epidemic
in the United States. Annu Rev Med 57, 181–194.
2 Chappell KJ, Stoermer MJ, Fairlie DP & Young PR
(2007) Generation and characterization of proteolyti-
cally active and highly stable truncated and full-length
recombinant WestNilevirus NS3. Protein Expr Purif
53, 87–96.
3 Luo D, Xu T, Hunke C, Gruber G, Vasudevan SG &
Lescar J (2008) Crystal structure ofthe NS3 protease–
helicase from Dengue virus. J Virol 82, 173–183.
4 Aleshin AE, Shiryaev SA, Strongin AY & Liddington
RC (2007) Structural evidence for regulation and
specificity of flaviviral proteases and evolution of the
Flaviviridae fold. Protein Sci 16, 795–806.
5 Erbel P, Schiering N, D’Arcy A, Renatus M, Kroemer
M, Lim SP, Yin Z, Keller TH, Vasudevan SG &
Hommel U (2006) Structural basis for the activation of
flaviviral NS3 proteases from dengue andWest Nile
virus. Nat Struct Mol Biol 13, 372–373.
6 Robin G, Chappell K, Stoermer MJ, Hu S, Young PR,
Fairlie DP & Martin JL (2009) Structure ofWest Nile
virus NS3 protease: ligand stabilization ofthe catalytic
conformation. J Mol Biol 385, 1568–1577.
7 Radichev I, Shiryaev SA, Aleshin AE, Ratnikov BI,
Smith JW, Liddington RC & Strongin AY (2008) Struc-
ture-based mutagenesis identifies important novel deter-
X C. Su et al. NMR analysis oftheWestNilevirus protease
FEBS Journal 276 (2009) 4244–4255 ª 2009 The Authors Journal compilation ª 2009 FEBS 4253
[...].. .NMR analysis oftheWestNilevirusprotease 8 9 10 11 12 13 14 15 16 17 18 X.-C Su et al minants ofthe NS2B cofactor oftheWestNilevirus two-component NS2B–NS3 proteinase J Gen Virol 89, 636–641 Chappell KJ, Stoermer MJ, Fairlie DP & Young PR (2008) Mutagenesis oftheWestNilevirus NS2B cofactor domain reveals two regions essential for protease activity J Gen Virol... effect of increasing concentrations of 2 on theNMR spectrum of WNV NS2B–NS3pro (K96A) Fig S3 15N-HSQC spectra of combinatorially 15Nlabelled samples of WNV NS2B–NS3pro in the presence of 1 Fig S4 800 MHz 1D 1H NMR spectra ofthe compounds 2 and 3 in the absence and presence of WNV NS2B–NS3pro(K96A) in D2O solution containing 1.5% d6-dimethylsulfoxide Fig S5 Superimposition of 15N-HSQC spectra of 0.3... Identification and biochemical characterization of small molecule inhibitorsofWestNileVirus serine protease by a high throughput screen Antimicrob Agents Chemother 52, 3385–3393 Johnston PA, Phillips J, Shun TY, Shinde S, Lazo JS, Huryn DM, Myers MC, Ratnikov BI, Smith JW, Su Y et al (2007) HTS identifies novel and specific uncompetitive inhibitorsofthe two-component NS2B–NS3 proteinase ofWestNile virus. .. high-throughput NMR studies Angew Chem Int Ed 46, 3356–3358 33 Melino S, Fucito S, Campagna A, Wrubl F, Gamarnik A, Cicero DO & Paci M (2006) The active essential CFNS3d protein complex – a new perspective for the structural and kinetic characterization ofthe NS2B– NS3pro complex of dengue virus FEBS J 273, 3650– 3662 34 Ekonomiuk D & Caflisch A (2009) Activation oftheWestNilevirus NS3 protease: molecular. .. protein synthesis: strategies for high-throughput NMR studies of proteins and protein–ligand complexes FEBS J 273, 4154–4159 Supporting information The following supplementary material is available: Fig S1 Assigned 15N-HSQC spectra of 0.9 mm solutions of 15N-labelled WNV NS2B–NS3pro(K96A) at 25 °C, pH 7.0, in the presence of 3 mm 2 or 3 Fig S2 Selected spectral region from 15N-HSQC spectra showing the effect... spectra of 0.3 mm WNV NS2B–NS3pro(K96A) in the presence of 0.5 mm 3 or 0.2 mm 3 + 0.4 mm Bz-nKRR-H Fig S6 Superimposition of 15N-HSQC spectra of 0.1 mm solutions of selectively 15N-Gly labelled WNV NS2B–NS3pro(G151A) in the absence and presence of 0.2 mm 3 Table S1 PCR primers used in this study to produce different variants of WNV NS2B–NS3pro This supplementary material can be found in the online article... Young PR & Fairlie DP (2001) Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, andinhibitors J Biol Chem 276, 45762–45771 Ekonomiuk D, Su XC, Ozawa K, Bodenreider C, Lim SP, Yin Z, Keller TH, Beer D, Patel V, Otting G et al (2009) Discovery of a non-peptidic inhibitor ofWestNilevirus NS3 protease by high-throughput docking... NE (2005) Cell-free in vitro protein synthesis in an autoinduction system for NMR studies of protein–protein interactions J Biomol NMR 32, 235–241 41 Wu PSC, Ozawa K, Jergic S, Su XC, Dixon NE & Otting G (2006) Amino-acid type identification in 15 N-HSQC spectra by combinatorial selective 15N-labelling J Biomol NMR 34, 13–21 NMR analysis oftheWestNilevirusprotease 42 Ozawa K, Wu PSC, Dixon NE &... Otting G (2004) Optimization of an Escherichia coli system for cell-free synthesis of selectively 15 N-labelled proteins for rapid analysis by NMR spectroscopy Eur J Biochem 271, 4084–4093 22 Shiryaev SA, Ratnikov BI, Chekanov AV, Sikora S, Rozanov DV, Godzik A, Wang J, Smith JW, Huang Z, Lindberg I et al (2006) Cleavage targets andthe d-arginine-based inhibitorsoftheWestNilevirus NS3 processing proteinase... & Strongin AY (2007) Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at theNS2B–NS3 junction region Protein Expr Purif 52, 334–339 24 Yin Z, Patel SJ, Wang WL, Chan WL, Rao KRR, Wang G, Ngew X, Patel V, Beer D, Knox JE et al (2006) Peptide inhibitorsof dengue virus NS3 protease Part 2: SAR studyof tetrapeptide aldehyde inhibitors Bioorg Med Chem Lett . NMR study of complexes between low molecular mass
inhibitors and the West Nile virus NS2B–NS3 protease
Xun-Cheng Su
1
, Kiyoshi. whereas the 3D NMR experi-
ments of the complexes with 2 and 3 employed the
WNV NS2B–NS3pro(K96A) mutant. The resonance
assignments of the complexes with 1 and